This work describes a novel plasmid encoding resistance to lincomycin in a staphylococcal isolate associated with mastitis infection from dairy cows. The cryptic plasmid pBMSa1 (2750 bp) of Staphylococcus aureus SA35 was subcloned and sequenced. Two ORFs (ORF1 and ORF2) were identified, and their putative transcription initiation and Shine-Dalgarno sequence were localized. ORF1 encodes a 334-residue protein almost identical to the putative Rep proteins of previously sequenced S. aureus rolling-circle-replicating plasmids. ORF2 encodes a 162-amino acid protein sharing a high degree of homology with LinA proteins (lincosamide O-nucleotidyltransferases) described in a variety of S. aureus strains. Intracellular single-stranded pBMSa1 DNA replicating intermediaries were detected, suggesting replication via the rolling-circle mechanism. A putative double-strand origin with significant homology to that of pC194 and a ssoA-type single-strand origin homologous sequence were also identified.